Methods of reducing density-dependent GC bias in amplification

ABSTRACT

The invention provides methods for amplifying nucleic acids, particularly methods for reducing density-dependent GC bias and for reducing nucleic acid damage in a bridge amplification of a nucleic acid template. The invention also provides methods for evaluating the effect of reagents and/or additives on nucleic acid damage during bridge amplification of nucleic acid template strands. The methods are suited to solid phase amplification, for example, utilizing flow cells.

FIELD OF THE INVENTION

The invention relates to methods of amplification of polynucleotidesequences and in particular relates to methods for amplification ofpolynucleotide sequences to minimize sequence specific biases. Themethods according to the present invention are suited to solid phaseamplification, for example, utilizing flow cells.

BACKGROUND

The Polymerase Chain Reaction or PCR (Saiki et al. (1985) Science230:1350) has become a standard molecular biology technique which allowsfor amplification of nucleic acid molecules. This in-vitro method is apowerful tool both for the detection and analysis of small quantities ofnucleic acids and other recombinant nucleic acid technologies.

Briefly, PCR typically utilizes a number of components: a target nucleicacid molecule, a molar excess of a forward and reverse primer which bindto the target nucleic acid molecule, deoxyribonucleoside triphosphates(dATP, dTTP, dCTP and dGTP) and a polymerase enzyme.

The PCR reaction is a DNA synthesis reaction that depends on theextension of the forward and reverse primers annealed to oppositestrands of a dsDNA template that has been denatured (melted apart) athigh temperature (90° C. to 100° C.). Using repeated melting, annealingand extension steps usually carried out at differing temperatures,copies of the original template DNA are generated.

Amplification of template sequences by PCR typically draws on knowledgeof the template sequence to be amplified such that primers can bespecifically annealed to the template. The use of multiple differentprimer pairs to simultaneously amplify different regions of the sampleis known as multiplex PCR, and suffers from numerous limitations,including high levels of primer dimerization, and the loss of samplerepresentation due to the different amplification efficiencies of thedifferent regions.

For the multiplex analysis of large numbers of target fragments, it isoften desirable to perform a simultaneous amplification reaction for allthe targets in the mixture, using a single pair of primers for all thetargets. In certain embodiments, one or more of the primers may beimmobilized on a solid support. Such universal amplification reactionsare described more fully in U.S. Patent App. Pub. No. 2005/0100900, theentire disclosure of which is incorporated herein by reference.Isothermal amplification methods for nucleic acid amplification aredescribed in U.S. Patent App. Pub. No. 2008/0009420, the entiredisclosure of which is incorporated herein by reference. The methodsinvolved may rely on the attachment of universal adapter regions, whichallows amplification of all nucleic acid templates from a single pair ofprimers.

The universal amplification reaction can still suffer from limitationsin amplification efficiency related to the sequences of the templates.One manifestation of this limitation is that the mass or size ofdifferent nucleic acid clusters varies in a sequence dependent manner.For example, the AT rich clusters can gain more mass or become largerthan the GC rich clusters. As a result, analysis of different clustersmay lead to bias. For example, in applications where clusters areanalyzed using sequencing by synthesis techniques, the GC rich clustersmay appear smaller or dimmer such that the clusters are detected lessefficiently. This results in lower representation of sequence data forGC rich clusters than the brighter (more intense) and larger AT richclusters. This can result in lower representation and less accuratesequence determination for the GC rich templates, an effect which may betermed GC bias.

The presence of sequence specific bias during amplification gives riseto difficulties determining the sequence of certain regions of thegenome, for example GC rich regions such as CpG islands in promoterregions. The resulting lack of sequence representation in the data fromclusters of different GC composition translates into data analysisproblems such as increases in the number of gaps in the analyzedsequence; a yield of shorter contigs, giving rise to a lower quality denovo assembly; identifying SNPs less accurately in GC rich regions dueto low coverage of these regions; and a need for increased coverage tosequence a genome, thereby increasing the cost of sequencing genomes.

The problem of bias may be more acute when the density of clusters onthe solid support is high. In certain situations, as the clusters grow,the amplification primers on the solid support are all extended, andhence adjacent clusters can not expand over the top of each other due tothe lack of available amplification primers. The over-amplification ofAT rich sequences causes rapid consumption of the primers on thesurface, and hence reduces the ability of the GC rich sequences toamplify.

In particular embodiments, the methods and compositions presented hereinare aimed at reducing density-dependent GC bias in isothermal acidamplification reactions.

One aspect of the invention provides a method for reducingdensity-dependent GC bias and/or nucleic acid damage in bridgeamplification of a double stranded nucleic acid template on a surfacecomprising:

-   -   a. denaturing the nucleic acid template with a first solution to        produce single stranded nucleic acid template strands;    -   b. optionally replacing the first solution with a second        solution;    -   c. annealing the single stranded nucleic acid template strands        to oligonucleotide primers bound to the surface; and    -   d. replacing the first or second solution with a solution        comprising a polymerase, whereby the oligonucleotide primers        bound to the surface are fully extended;

wherein at least one of the solutions comprises at least one additiveand density-dependent GC bias and/or nucleic acid damage in theisothermal amplification of the double stranded nucleic acid template isreduced.

One type of primer may be on the surface and the other primer insolution.

Preferably, at least one of the first and second solutions comprises atleast one additive.

In one embodiment, only the solution comprising a polymerase comprisesan additive, such as a high concentration of betaine.

The bridge amplification may be a substantially isothermal bridgeamplification.

The nucleic acid may comprise DNA or RNA.

Optionally, the surface is a flow cell surface.

In one embodiment, the surface may comprise a bead.

Replacing the first or second solution may comprise mixing solutionsand/or gradually replacing a solution via a continuous gradient.

Preferably, a cluster of identical nucleic acid strands is generated.

In a preferred embodiment, the first solution comprises formamide.

The additive may comprise a chelating agent.

The additive may comprise a mixture of different dNTPs and/or rNTPs.

The additive may comprise a single type of dNTP and/or a single type ofrNTP.

The additive may comprise at least one citrate.

The at least one citrate may be selected from: monosodium citrate,disodium citrate, trisodium citrate, and potassium citrate.

The additive may comprise EDTA.

The additive may comprise betaine.

The additive may comprise DMSO.

Preferably, the second solution comprises a low ionic strength solution.

In one embodiment, the second solution comprises water.

Preferably, the second solution comprises less than about 100 mM ofsalt.

Preferably, the second solution comprises less than about 80 mM of salt;more preferably less than about 40 mM of salt; even more preferably lessthan about 20 mM of salt, more preferably less than about 10 mM of salt.Even more preferably, the second solution comprises less than about 5 mMof salt.

In one embodiment, the second solution is substantially free from anysalt.

In one embodiment, the second solution comprises a pre-mix solution.

The pre-mix solution may comprise one or more salts and/or buffers.

The pre-mix solution may comprise DMSO and/or betaine.

In one embodiment, the second solution does not comprise an additive.

Preferably, the solution comprising the polymerase comprises a mixtureof different dNTPs or rNTPs.

In an embodiment in which the nucleic acid comprises DNA, the solutioncomprising the polymerase comprises dNTPs.

In an embodiment in which the nucleic acid comprises RNA, the solutioncomprising the polymerase comprises rNTPs.

In an embodiment in which the nucleic acid comprises DNA, the polymerasepreferably comprises a DNA polymerase.

Optionally, the DNA polymerase comprises a Bst polymerase.

In an embodiment in which the nucleic acid comprises RNA, the polymerasepreferably comprises an RNA polymerase.

In an embodiment in which the nucleic acid comprises RNA, the solutioncomprising the polymerase preferably comprises a mixture of rNTPs.

Optionally, the solution comprising the polymerase comprises at leastone additive selected from one or more of the following: EDTA, a mixtureof dNTPs, a single type of dNTP, a mixture of rNTPs, a single type ofrNTP, a citrate, a citrate salt, monosodium citrate, disodium citrate,trisodium citrate, potassium citrate, betaine, DMSO.

In one embodiment additive comprises a high concentration of betaine.

Preferably, the solution comprising the polymerase does not compriseammonium sulphate.

Preferably, the solution comprising the polymerase comprises less thanabout 100 mM of any salt.

Preferably, the solution comprising the polymerase comprises less thanabout 80 mM of salt; more preferably less than about 40 mM of salt; evenmore preferably less than about 20 mM of salt, more preferably less thanabout 10 mM of salt. Even more preferably, the solution comprising thepolymerase comprises less than about 5 mM of salt.

In one embodiment, the solution comprising the polymerase issubstantially free from any salt.

In one embodiment, the solution comprising the polymerase comprises oneor more salts and/or buffers.

In one embodiment, the solution comprising the polymerase comprises DMSOand/or betaine.

Optionally, the additive may comprise a mixture of ddNTPs or a singletype of ddNTP.

Steps (a) to (d) of the method are preferably performed two or moretimes.

The steps may be performed about 35 times, or less.

In one embodiment, the steps are performed more than 35 times. In oneembodiment, the steps are performed around 50 times.

The steps may be performed at about 60° C., or less.

The steps may be performed at about 50° C. or less. The steps may beperformed at about 40° C. or less. The steps may be performed at about30° C. or less.

The steps may be performed while the temperature is being cycled betweentwo or more different temperatures.

Another aspect of the invention provides a method for evaluating damageof nucleic acid strands by a reagent and/or additive comprising;

attaching the nucleic acid strands to a surface;

introducing the reagent and/or additive to the nucleic acid strandsattached to the surface;

visualising the nucleic acid strands to detect damage thereof by thereagent and/or additive.

The amount of damage may be quantified.

Preferably, the amount of damage is evaluated by detecting the number ofundamaged molecules and comparing the number to the number of undamagedmolecules obtained in a control method.

The control method lacks the introduction of the reagent and/oradditive.

The step of attaching the nucleic acid strands to a surface optionallycomprises seeding the surface with the nucleic acid strands wherebysingle stranded nucleic acid strands anneal to oligonucleotide primersbound to the surface.

The oligonucleotide primers may be extended using a polymerase.

Optionally, the method includes the step of denaturing single strandednucleic acid strands, whereby single stranded nucleic acid moleculescovalently bound to the oligonucleotide primers bound to the flow cellsurface are produced.

Optionally, the method includes the step of performing a mockamplification method on the single stranded nucleic acid moleculescovalently bound to the oligonucleotide primers bound to the flow cellsurface

The method may comprise cycling the reagent and/or additive, pumping thereagent and/or additive substantially continuously to the nucleic acidstrands

In another embodiment, the step of introducing the reagent and/oradditive comprises substantially static incubation with the nucleic acidstrands.

Optionally, the method includes the step of performing an amplificationmethod on the single stranded nucleic acid molecules covalently bound tothe oligonucleotide primers and visualizing clusters of identicalnucleic acid strands.

Preferably, the amplification is carried out after the nucleic aciddamaging treatment.

The amplification method may comprise bridge amplification.

Clusters of identical nucleic acid strands may be stained with a bindingdye and imaged.

Advantageously, the number of clusters of identical nucleic acid strandsis inversely proportional to the amount of damage.

In one embodiment, the step of extending the oligonucleotide primersusing a polymerase may be performed after the step of performing themock isothermal amplification.

The reagent may comprise a cluster amplification reagent.

The reagent may comprise a damaging agent of physical or chemical orphysical nature.

The additive may comprise one or more of: a chelating agent, EDTA, amixture of dNTPs, a single type of dNTP, a citrate, a citrate salt,monosodium citrate, disodium citrate, trisodium citrate, potassiumcitrate, betaine, DMSO.

Optionally, the method is for evaluating the effect of clusteramplification reagents and/or additives on nucleic acid damage.

The method may be for evaluating the effect of chemical or physicalagents, on nucleic acid damage.

The nucleic acid may comprise DNA or RNA.

In one embodiment the nucleic acid comprises single stranded DNA.

In one embodiment, the nucleic acid comprises double stranded DNA.

The method may include a preliminary step of denaturing double strandedDNA to provide single stranded template strands.

Optionally, the amplification method comprises substantially isothermalbridge amplification.

The mock amplification method may comprise;

-   -   i. adding a first solution comprising a first additive to the        single stranded nucleic acid molecules covalently bound to the        oligonucleotide primers bound to the flow cell surface;    -   ii. replacing the first solution with a second solution        comprising a second additive; and    -   iii. replacing the second solution with a third solution that        does not comprise a polymerase.    -   wherein density-dependent GC bias and/or nucleic acid damage in        the isothermal amplification of the double stranded nucleic acid        template is reduced.

Optionally, the method is for evaluating nucleic acid damage duringbridge amplification.

The mock amplification may be a substantially isothermal mockamplification.

In one embodiment, the step of performing an amplification methodcomprises

-   -   a. denaturing a nucleic acid template with a first solution to        produce single stranded nucleic acid template strands;    -   b. optionally replacing the first solution with a second        solution;    -   c. annealing the single stranded nucleic acid template strands        to oligonucleotide primers bound to the surface; and    -   d. replacing the first or second solution with a solution        comprising a polymerase, whereby the oligonucleotide primers        bound to the surface are fully extended;

wherein at least one of the first and second solutions comprises atleast one additive and density-dependent GC bias and/or nucleic aciddamage in the isothermal amplification of the double stranded nucleicacid template is reduced.

The bridge amplification may be a substantially isothermal bridgeamplification.

The nucleic acid may comprise DNA or RNA.

Optionally, the surface is a flow cell surface.

In one embodiment, the surface may comprise a bead.

Replacing the first or second solution may comprise mixing solutionsand/or gradually replacing a solution via a continuous gradient.

Preferably, a cluster of identical nucleic acid strands is generated.

In a preferred embodiment, the first solution comprises formamide.

The additive may comprise a chelating agent.

The additive may comprise a mixture of different dNTPs and/or rNTPs.

The additive may comprise a single type of dNTP and/or a single type ofrNTP.

The additive may comprise at least one citrate.

The at least one citrate may be selected from: monosodium citrate,disodium citrate, trisodium citrate, and potassium citrate.

The additive may comprise EDTA.

The additive may comprise betaine.

The additive may comprise DMSO.

Preferably, the second solution comprises a low ionic strength solution.

In one embodiment, the second solution comprises water.

Preferably, the second solution comprises less than about 100 mM ofsalt.

Preferably, the second solution comprises less than about 80 mM of salt;more preferably less than about 40 mM of salt; even more preferably lessthan about 20 mM of salt, more preferably less than about 10 mM of salt.Even more preferably, the second solution comprises less than about 5 mMof salt.

In one embodiment, the second solution is substantially free from anysalt.

In one embodiment, the second solution comprises a pre-mix solution.

The pre-mix solution may comprise one or more salts and/or buffers.

The pre-mix solution may comprise DMSO and/or betaine.

In one embodiment, the second solution does not comprise an additive.

Preferably, the solution comprising the polymerase comprises a mixtureof different dNTPs or rNTPs.

In an embodiment in which the nucleic acid comprises DNA, the solutioncomprising the polymerase comprises dNTPs.

In an embodiment in which the nucleic acid comprises RNA, the solutioncomprising the polymerase comprises rNTPs.

In an embodiment in which the nucleic acid comprises DNA, the polymerasepreferably comprises a DNA polymerase.

Optionally, the DNA polymerase comprises a Bst polymerase.

In an embodiment in which the nucleic acid comprises RNA, the polymerasepreferably comprises an RNA polymerase.

In an embodiment in which the nucleic acid comprises RNA, the solutioncomprising the polymerase preferably comprises a mixture of rNTPs.

Optionally, the solution comprising the polymerase comprises at leastone additive selected from one or more of the following: EDTA, a mixtureof dNTPs, a single type of dNTP, a mixture of rNTPs, a single type ofrNTP, a citrate, a citrate salt, monosodium citrate, disodium citrate,trisodium citrate, potassium citrate, betaine, DMSO.

In one embodiment additive comprises a high concentration of betaine.

Preferably, the solution comprising the polymerase does not compriseammonium sulphate.

Preferably, the solution comprising the polymerase comprises less thanabout 100 mM of any salt.

Preferably, the solution comprising the polymerase comprises less thanabout 80 mM of salt; more preferably less than about 40 mM of salt; evenmore preferably less than about 20 mM of salt, more preferably less thanabout 10 mM of salt. Even more preferably, the solution comprising thepolymerase comprises less than about 5 mM of salt.

In one embodiment, the solution comprising the polymerase issubstantially free from any salt.

In one embodiment, the solution comprising the polymerase comprises oneor more salts and/or buffers.

In one embodiment, the solution comprising the polymerase comprises DMSOand/or betaine.

Optionally, the additive may comprise a mixture of ddNTPs or a singletype of ddNTP.

Steps of the method are preferably performed two or more times.

The steps may be performed about 35 times, or less. In one embodiment,the steps are performed more than 35 time. In one embodiment, the stepsare performed around 50 times.

The steps may be performed at about 60° C., or less. The steps may beperformed at about 50° C. or less. The steps may be performed at about40° C. or less. The steps may be performed at about 30° C. or less.

In one embodiment, the first solution comprises formamide and at leastone additive, the second solution comprises water and no additive, andthe solution comprising the polymerase comprises no additive.

Another aspect of the invention provides a system for bridgeamplification comprising apparatus having at least one inlet, and atleast one outlet; and means for reducing density-dependent GC biasand/or nucleic acid damage in a bridge amplification of a doublestranded nucleic acid template on a surface.

The system preferably comprises control means for coordinating the stepsof the method.

The apparatus may comprise means for immobilizing primers on a surface.

Optionally, the apparatus comprises a flow cell and solutions areapplied through the inlet and removed through the outlet by a process ofsolution exchange.

Optionally, the system comprises detection means for detecting afluorescent signal.

Yet another aspect of the invention provides a system for evaluatingnucleic acid damage comprising apparatus having at least one inlet, andat least one outlet; and means for evaluating nucleic acid damage ofnucleic acid strands on a surface.

Preferably, the surface comprises a flow cell surface.

In one embodiment the system is for evaluating nucleic acid damageduring bridge amplification.

Yet another aspect of the invention provides a kit for reducingdensity-dependent GC bias and/or nucleic acid damage in a bridgeamplification of a double stranded nucleic acid template on a surfacecomprising;

a first solution for producing single stranded nucleic acid templatestrands; at least one additive; and a polymerase.

Optionally the bridge amplification comprises substantially isothermalbridge amplification.

Preferably, the at least one additive comprises: a chelating agent,EDTA, a mixture of dNTPs, a single type of dNTP, a mixture of rNTPs, asingle type of rNTP, a citrate, a citrate salt, monosodium citrate,disodium citrate, trisodium citrate, potassium citrate, betaine and/orDMSO.

The first solution may comprise formamide.

Optionally, the kit further comprises a second solution comprising apre-mix and/or water.

Yet another aspect of the invention provides a kit for evaluatingnucleic acid damage of nucleic acid template strands on a surfacecomprising at least one reagent and/or additive and means forvisualising the nucleic acid strands.

Means for visualising the nucleic acid strands may comprise a dye.

Optionally, the kit or system is for evaluating the effect of clusteramplification reagents on nucleic acid damage.

The kit may comprise primers and/or instructions for performing themethod.

BRIEF DESCRIPTION

A method for reducing density-dependent GC bias in an isothermal bridgeamplification of a double stranded DNA (dsDNA) template on a flow cellsurface is provided, comprising: a) denaturing the dsDNA template with afirst solution comprising a first additive to produce single strandedDNA (ssDNA) template strands; b) replacing the first solution with asecond solution which may comprise a second additive, whereby the ssDNAtemplate strands anneal to oligonucleotide primers bound to the flowcell surface; and c) replacing the second solution with a third solutioncomprising a DNA polymerase, whereby the oligonucleotide primers boundto the flow cell surface are fully extended; wherein density-dependentGC bias in the isothermal amplification of the dsDNA template isreduced. In one embodiment, a cluster of identical DNA strands isgenerated. In another embodiment, the first solution comprisesformamide. In one embodiment, the first solution further comprises EDTA.In one embodiment, the first solution comprises formamide together witha high concentration of betaine. In some embodiments, the first additiveis a mixture of different dNTPs or is a single type of dNTP. In afurther embodiment, the first additive is trisodium citrate. In stillfurther embodiments, the second additive is a mixture of differentdNTPs. In some embodiments, the second solution further comprises apre-mix solution, particularly wherein the pre-mix solution comprisesone or more salts and one or more buffers, more particularly wherein thepre-mix solution comprises DMSO and/or Betaine. In another embodiment,the second solution comprises water. In yet another embodiment, the DNApolymerase is a Bst DNA polymerase. In further embodiments, the thirdsolution further comprises a mixture of different dNTPs. In stillfurther embodiments, the third solution comprises one or more salts andone or more buffers, particularly wherein the pre-mix solution comprisesDMSO and/or Betaine. In another embodiment, the third solution does notcomprise ammonium sulfate. In additional embodiments, steps (a) to (c)are performed two or more times, particularly wherein steps (a) to (c)are performed about 35 times, and more particularly wherein steps (a) to(c) are performed at about 60° C.

In some embodiments, the temperature at which steps (a) to (c) areperformed may be adjusted according to the optimum temperature for thetype of DNA polymerase used.

A method for evaluating the effect of cluster amplification reagentsand/or additives on DNA damage during isothermal bridge amplification ofssDNA template strands on a flow cell surface is also provided,comprising: a) seeding the flow cell surface with the ssDNA templatestrands, whereby the ssDNA template strands anneal to oligonucleotideprimers bound to the flow cell surface; b) extending the oligonucleotideprimers using a DNA polymerase; c) denaturing the dsDNA templatestrands, whereby ssDNA molecules covalently bound to the oligonucleotideprimers bound to the flow cell surface are produced; d) performing amock isothermal amplification method on the ssDNA molecules covalentlybound to the oligonucleotide primers bound to the flow cell surface,comprising cycling cluster amplification reagents and/or additives; e)performing an isothermal amplification method on the ssDNA moleculescovalently bound to the oligonucleotide primers bound to the flow cellsurface; and f) visualizing clusters of identical DNA strands, whereinclusters of identical DNA strands are stained with a DNA binding dye andimaged; wherein the number of clusters of identical DNA strands isinversely proportional to the amount of DNA damage caused by the clusteramplification reagents and/or additives. In one embodiment, the mockisothermal amplification method comprises: i) adding a first solutioncomprising a first additive to the ssDNA molecules covalently bound tothe oligonucleotide primers bound to the flow cell surface; ii)replacing the first solution with a second solution comprising a secondadditive; and iii) replacing the second solution with a third solutionthat does not comprise a DNA polymerase. In an alternative embodiment,step (d) may be replaced with an alternative step (d) of pumpingamplification reagents and/or additives substantially continuously tothe ssDNA molecules covalently bound to the oligonucleotide primersbound to the flow cell surface.

In one embodiment, the first solution, second solution and/or thirdsolutions used in step (d) do not comprise additives.

In another embodiment, the isothermal amplification method comprises: i)adding a first solution comprising a first additive to the ssDNAmolecules covalently bound to the oligonucleotide primers bound to theflow cell surface; ii) replacing the first solution with a secondsolution comprising a second additive; and iii) replacing the secondsolution with a third solution comprising a DNA polymerase.

In one embodiment, the first solution, second solution and/or thirdsolutions used in the isothermal amplification step (e) do not compriseadditives.

In some embodiments, the first solution comprises formamide. The firstadditive may comprise one or more of the following: a chelating agent,EDTA, a mixture of dNTPs, a single type of dNTP, a mixture of rNTPs, asingle type of rNTP, a a citrate, a citrate salt, sodium citrate,disodium citrate, trisodium citrate, potassium citrate, betaine, DMSO.

In one embodiment, the first additive comprises EDTA. In someembodiments, the first additive is a mixture of different dNTPs or thefirst additive is a single type of dNTP. In a further embodiment, thefirst additive is trisodium citrate. In still further embodiments, thesecond additive is a mixture of different dNTPs. In some embodiments,the second solution comprises a pre-mix solution, particularly whereinthe pre-mix solution comprises one or more salts and one or morebuffers, more particularly wherein the pre-mix solution comprises DMSOand/or Betaine. In another embodiment, the second solution compriseswater.

The second additive may comprise one or more of the following: achelating agent, EDTA, a mixture of dNTPs, a single type of dNTP, acitrate, a citrate salt, monosodium citrate, disodium citrate, trisodiumcitrate, potassium citrate, betaine, DMSO.

In yet another embodiment, the DNA polymerase is a Bst DNA polymerase.In further embodiments, the third solution further comprises a mixtureof different dNTPs. In still further embodiments, the third solutioncomprises one or more salts and one or more buffers, particularlywherein the pre-mix solution comprises DMSO and/or Betaine. In anotherembodiment, the third solution does not comprise ammonium sulphate orother salt. In additional embodiments, steps (a) to (d) are performedtwo or more times, particularly wherein steps (a) to (d) are performedabout 26 times, more particularly wherein steps (a) to (d) are performedabout 35 times or lower, and even more particularly wherein steps (a) to(d) are performed at about 60° C.

In some embodiments, the temperature at which steps (a) to (d) areperformed may be adjusted according to the optimum temperature for thetype of DNA polymerase used in the amplification process.

A method for reducing DNA damage in an isothermal bridge amplificationof a dsDNA template on a flow cell surface is also provided, comprising:a) denaturing the dsDNA template with a first solution comprisingformamide to produce single stranded DNA (ssDNA) template strands; b)replacing the first solution with a second solution comprising dNTPs,whereby the ssDNA template strands anneal to oligonucleotide primersbound to the flow cell surface; and c) replacing the second solutionwith a third solution comprising a DNA polymerase, whereby theoligonucleotide primers bound to the flow cell surface are fullyextended; wherein DNA damage in the isothermal amplification of thedsDNA template is reduced.

FIG. 1 illustrates a flow diagram of an example of an isothermal bridgeamplification protocol for cluster amplification on a flow cell surface;

FIGS. 2A and 2B show a plot of cluster size and a plot of clusterintensity, respectively, of clusters generated from monotemplates with a400 bp insert with different GC composition;

FIG. 3 shows a screenshot of a cluster image showing GC-rich clustersand AT-rich clusters, wherein four monotemplates were seeded atrelatively low density;

FIG. 4 illustrates a flow diagram of an example of a DNA damage assayfor evaluating the effect of cluster amplification reagents and/or theaddition of additives on cluster generation during isothermal bridgeamplification;

FIG. 5 shows pictorially the steps of the DNA damage assay of FIG. 4;

FIG. 6 shows a bar graph of the percentage of non-damaged DNA moleculesin samples treated with formamide, pre-mix, and/or pre-mix plus dNTPs,compared to a lane treated with wash buffer only and evaluated using theDNA damage assay of FIG. 4;

FIG. 7 shows a bar graph of the percentage of non-damaged DNA moleculesin samples treated with formamide±dNTPs, pre-mix±dNTPs, or water±dNTPsand evaluated using the DNA damage assay of FIG. 4;

FIG. 8 shows a dose response curve of the effect of dNTP concentrationin formamide on cluster number during mock isothermal amplification;

FIG. 9 shows a bar graph of the effectiveness of each dNTP to preventDNA damage when used as a formamide additive;

FIG. 10 shows panels of clusters stained with SYBR® Green generated byisothermal amplification using formamide with and without added dNTPs;

FIG. 11 shows a data table of sequencing metrics for a sequencing runevaluating the “dNTPs method” and the “water method”;

FIGS. 12A and 12B show curves of GC bias and curves of GC biasnormalized to low density lane 1, respectively, of the sequencing rundescribed with reference to FIG. 11;

FIG. 13A shows a summary schematic diagram of bridge amplificationsolutions according to the methods of the invention; and

FIG. 13B shows a summary schematic diagram of bridge amplificationsolutions according to a standard method.

FIG. 14 shows the effect of various formamide additives on DNA damage byisothermal cluster amplification reagents (FC61WEBAAXX, exp 12374).CT180 was seeded at 1 pM. 1^(st) strand extension was done using Taq DNApolymerase followed by NaOH denaturation. Single molecules were thentreated with 26 cycles of “mock isothermal amplification (28 ulformamide, 28 ul H2O, 36 ul premix with dNTPs. The control lane wastreated with wash buffer for the entire duration). WB=wash buffer.dNTPs=200 uM of each nucleotide. AMP=adenosine monophosphate at 800 uMfinal concentration. 2M betaine in formamide=60% formamide containingbetaine at final concentration of 2M and water. 1M betaine informamide=80% formamide containing betaine at final concentration of 1Mand water. 0.5M betaine in formamide=90% formamide containing betaine atfinal concentration of 0.5M and water.

FIG. 15. Protective effect against DNA damage of each individualcomponent of wash buffer (FC62263AAXX exp 12402). CT180 was seeded at0.6 pM. 1^(st) strand extension was done using Taq DNA polymerasefollowed by NaOH denaturation. Single molecules were then treated with26 cycles of “mock isothermal amplification (28 ul formamide withvarious additives, 28 ul H2O, 36 ul premix with dNTPs.) Lane 1 acted asa control and was treated with wash buffer for the entire duration ofthe treatment. 2^(nd) and 3^(rd) solutions were 28 ul of H2O and 36 ulof pre-mix/dNTPs respectively for all lanes except for lane 1 in whichwash buffer was pumped instead. WB=wash buffer. dNTPs=200 uM of eachnucleotide. Each additive was added to a concentration that is identicalto that which would be reached when adding 10% of wash buffer (4.5 mMNaCl, 0.45 mM Na Citrate, 0.01% tween-20).

FIG. 16. Effect of the concentration of sodium Citrate in formamide onDNA damage (FC62272AAXX, exp 12411). CT180 was seeded at 0.6 pM. 1^(st)strand extension was done using Taq DNA polymerase followed by NaOHdenaturation. Single molecules were then treated with 26 cycles of “mockisothermal amplification (28 ul formamide, 28 ul H2O and 36 ul premixwith dNTPs). One lane was used as a control and was treated by pumpingwash buffer for the entire duration of the treatment. At the end of thetreatment, cluster amplification was performed using 26 cycles ofisothermal amplification (28 ul formamide+64 ul Bst mix). Clusters werethen stained with sybr green and three tiles per lane were imaged uisnga microscope and a camera. Clusters numbers were determined using asoftware called Firecrest.

FIGS. 17A and 17B relate to the effect of the presence in formamide ofthe chelating molecule EDTA and of the ion magnesium on DNA damage(FC6254LAAXX, exp 12473). FIG. 17A depicts an experiment design. CT180was seeded at 1 pM. 1^(st) strand extension was done using Taq DNApolymerase followed by NaOH denaturation. Single molecules were thentreated with 26 cycles of “mock isothermal amplification (28 ulformamide, 28 ul H2O, 36 ul premix with dNTPs except for lane 1 whichwas treated with wash buffer for the entire duration. The pre-mix waseither standard containing 10 mM ammonium sulfate or a pre-mix that wasammonium sulphate-free. EDTA (0.5M, pH 8.0) was added to formamide to afinal concentration of 1 mM. dNTPs were added to formamide to a finalconcentration of 200 uM of each nucleotide (800 uM total concentration).In lane 7, Mg sulphate was added to a final concentration of 3 mM to thedNTPs containing formamide solution. After bridge amplification,clusters were then stained with sybr green and three tiles per lane wereimaged using a microscope and a camera. Clusters numbers were determinedusing a software called Firecrest. FIG. 17B is a graph in which clusternumbers are plotted as a percentage of lane 1 (wash buffer treatedcontrol lane).

FIG. 18. Effect of increasing the concentration of ammonium sulphate inthe Bst mix on density-dependent GC bias. Run summary (A) and GC biascurves (B and C) from experiment 100526_EAS20_0170_FC61LRUAAXX. CT3576(standard human library, 300 bp insert average size) was seeded ateither low density (1 pM) or high density (5.5 pM). After standard firststrand extension with Phusion, cluster were amplified with 26 cycles ofisothermal amplification using 28 ul of formamide followed by 28 ul ofwater and 36 ul of Bst mix with different concentrations of ammoniumsulphate (ranging between the standard 10 mM concentration and up to 80mM). The bias curves in C were obtained from the curves in (B) bydividing each curve by the low density lane (lane 1) curve as a way offocusing exclusively on the density-dependent component of the GC biascreated during cluster amplification.

FIG. 19. Combined effect of dNTPs in formamide and lower ammoniumsulphate concentrations in Bst mix on density-dependent GC bias. Theexperiment design is shown in (A). After seeding a standard humanlibrary (CT3576) at high and low density and 1^(st) strand extensionwith Phusion, clusters were amplified using 26 cycles of isothermalamplification with the conditions shown in the table. After read 1preparation the flowcell was sequenced with a GAllx instrument. Thisparticular run was a paired end experiment using the 95G chemistrysequencing 36 bases in each read. Analysis was carried out with pipeline1.6. (B) shows the summary for read 1 whereas in C are shown thenormalised GC bias curves (obtained by dividing the original GC biascurves by the curve at low density (lane 1).

FIG. 20. Combining sodium citrate with ammonium-free Bst mix and top-upcycles. The experiment design is shown in (A). After seeding of thestandard human library (CT3576) at high and low density and 1^(st)strand extension with Phusion, clusters were amplified using 26 or 32cycles of isothermal amplification with the conditions shown in thetable. After read 1 preparation the flowcell was sequenced with a GAllxinstrument. This particular experiment was a paired end run using the95G chemistry sequencing 36 bases in each read. Analysis was carried outwith pipeline 1.8. In (B) is shown the summary for read 1 and thenormalised GC bias curves (obtained by dividing the original GC biascurves by the curve at low density lane 1) are shown in C. A directcomparison in terms of density-dependent GC bias of dNTPs in formamideand water and sodium citrate in formamide, both with an ammonium-freeBst mix is shown in D.

FIG. 21. Effect of sodium citrate concentration and betaine informamide. The experiment design is shown in (A). After seeding of astandard human library (CT3576) at high and low density and 1^(st)strand extension with Phusion, clusters were amplified using either 35cycles (with standard reagents, lanes 1 and 2) or 32 cycles ofisothermal (with an ammonium-free Bst mix, lanes 3 to 8). After read 1preparation the flowcell was sequenced with a GAllx instrument. Thisparticular experiment was a single read run using the 95G chemistrysequencing (36 cycles of SBS). Analysis was carried out with pipeline1.8. (B) shows the summary for read 1 whereas the normalised GC biascurves (obtained by dividing the original GC bias curves by the curve atlow density lane 1) are shown in C.

DEFINITIONS

As used herein, the following terms have the meanings indicated.

The terms “normalize” or “reduce sequence specific bias” (e.g., reducingdensity-dependent GC bias) when used in reference to the amplificationof nucleic acid templates, means to alter the ratio of molecules ofdifferent type obtained during an amplification process such that thenumber of molecules of a particular type in the population is made moreequal to the number of molecules of another type in the population. Thusfor an amplification reaction carried out on a population of nucleicacid templates of different sequence, to normalize the amplification canmean lowering any sequence specific biases which would otherwise resultin certain members of the population increasing in number more thanother members of the population. The normalization process can be usedto produce relative ratios of the fragments in the population that arethe same after the amplification as they were in the population beforeamplification. Thus for example a population comprising 1 millionmolecules of different sequence will contain, after amplification, onaverage the same number of copies of each of the 1 million fragmentswithout any specific biases for certain sequences. It will be understoodthat this is a statistical measure and that the absence of bias can bewithin an acceptable variance such as within 0.5, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45 or 50% variance in the number of copies for eachfragment. When carried out on a solid support to make nucleic acidclusters, the normalization of the amplification results in an array ofclusters with a similar number of molecules in each cluster, and thussimilar sizes and signal intensities.

The term “different” when used in reference to two or more nucleic acidsmeans that the two or more nucleic acids have nucleotide sequences thatare not the same. For example, two nucleic acids can differ due to onesequence being longer than the other and conversely one sequence beingshorter than the other. Two nucleic acids can differ in the content andorder of nucleotides in the sequence of one nucleic acid compared to theother nucleic acid, independent of any differences in sequence lengthbetween the two nucleic acids. The term can be used to describe nucleicacids whether they are referred to as copies, amplicons, templates,targets, primers, oligonucleotides, polynucleotides, or the like.

As described herein, nucleic acid templates containing a high level of Aand T bases typically amplify more efficiently than nucleic acidtemplates with a high level of G and C bases. Nucleic acid templateswith sequences containing a high level of A or T bases compared to thelevel of G or C bases are referred to throughout as AT rich templates ortemplates with high AT content. Accordingly, AT rich templates can haverelatively high levels of A bases, T bases or both A and T bases.Similarly, nucleic acid templates with sequences containing a high levelof G or C bases compared to the level of A or T bases are referred tothroughout as GC rich templates or templates with high GC content.Accordingly, GC rich templates can have relatively high levels of Gbases, C bases or both G and C bases. The terms GC rich and high GCcontent are used interchangeably. Similarly, the terms AT rich and highAT content are used interchangeably. The phrases GC rich and AT rich, asused herein, refer to a nucleic acid sequence having a relatively highnumber of G and/or C bases or A and/or T bases, respectively, in itssequence, or in a part or region of its sequence, relative to thesequence content contained within a control. In this case, the controlcan be similar nucleic acid sequences, genes, or the genomes from whichthe nucleic acid sequences originate. Generally, nucleic acid sequenceshaving greater than about 52% GC or AT content are considered GC rich orAT rich sequences. Optionally, the GC content or AT content is greaterthan 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%. Nucleic acid sequencescontaining discrete regions of high GC to AT content may also beconsidered GC rich or AT rich, respectively. The methods provided hereinnormalize the efficiencies or levels of amplification of templates withdifferent sequence, for example, with high AT and/or GC content.

The term “amplification cycle” refers to one or more steps of anamplification process that are sufficient to produce one or more copiesof a nucleic acid template. By way of example, an amplification cycleincludes providing one or more nucleic acid templates, denaturing thenucleic acid templates to produce single stranded nucleic acidtemplates, annealing one or more primers to the single stranded nucleicacid templates, and extending the primers to produce copies of thesingle stranded nucleic acid templates. As described herein, such cyclescan be repeated one or more times under conditions favoring AT rich orGC rich templates. Thus, a cycle of amplification can include a unit ofone or more steps that is repeated in a round of amplification.

As used throughout, the phrase “favoring AT rich templates” means thatthe efficiency of amplification of AT rich templates is not reduced orinhibited relative to non-AT rich templates. By way of example, understandard amplification conditions, AT rich templates amplify at a higherefficiency than GC rich templates. Thus, conditions favoring AT richtemplates include standard amplification conditions. As used throughout,the phrase standard amplification conditions means amplifying a nucleicacid sequence under conditions including all standard reagents andconditions necessary to carry out amplification. Standard amplificationconditions are known and described in, for example, Saiki et al.,Science, 230:1350 (1985).

As used herein, the phrase “favoring GC rich templates” means that theefficiency of amplification of GC rich templates is increased relativeto AT rich templates and/or the efficiency of amplification of AT richtemplates is reduced relative to GC rich templates.

The nucleotides used in the amplification process may be ribo- ordeoxyribo-nucleotides. The nucleotides used in the amplification may benucleotide 5′ polyphosphates, for example 5′triphosphates. Thenucleotides used in the amplification reaction may be the fournucleotide triphosphates typically found in native DNA: dATP, dGTP, dCTPand dTTP.

As used herein, the terms high, higher, increase(s), increased, orincreasing refer to any increase above a reference or control, unlessstated otherwise. The terms low, lower, decrease(s), decreased,decreasing, reduce(s), reduced, reducing or reduction refer to anydecrease below a reference or control, unless stated otherwise. By wayof example, a control includes control values or control levels, whichcan be values or levels prior to, or in the absence of, a stimulus. Acontrol or control value includes the level of efficiency ofamplification of nucleic acid sequences under standard amplificationconditions or can comprise a known value, level or standard. Thus, forexample, a higher or lower value (e.g., temperature or concentration) ascompared to a control refers to a value that is higher or lower than aknown or arbitrarily set value.

The term “isothermal” refers to thermodynamic processes in which thetemperature of a system remains constant: DT=0. This typically occurswhen a system is in contact with an outside thermal reservoir (forexample, heat baths and the like), and processes occur slowly enough toallow the system to continually adjust to the temperature of thereservoir through heat exchange.

The term “substantially isothermal” as used herein is intended to meanthat the system is maintained at essentially the same temperature. Theterm is also intended to capture minor deviations in temperature whichmight occur as the system equilibrates, for example when componentswhich are of lower or higher temperature are added to the system. Thusit is intended that the term includes minor deviations from thetemperature initially chosen to perform the method and those in therange of deviation of commercial thermostats. Particularly thetemperature deviation will be no more than about +/−2° C., moreparticularly no more than about +/−1° C., yet more particularly no morethan about +/−0.5° C., no more than about +/−0.25° C., no more thanabout +/−0.1° C. or no more than about +/−0.01° C.

The term “amplifying” as used herein is intended to mean the process ofincreasing the numbers of a template polynucleotide sequence byproducing one or more copies. Accordingly it will be clear that theamplification process can be either exponential or linear. Inexponential amplification the number of copies made of the templatepolynucleotide sequence increases at an exponential rate. For example,in an ideal PCR reaction with 30 cycles, 2 copies of template DNA willyield 2³⁰ or 1,073,741,824 copies. In linear amplification the number ofcopies made of the template polynucleotide sequences increases at alinear rate. For example, in an ideal 4-hour linear amplificationreaction whose copying rate is 2000 copies per minute, one molecule oftemplate DNA will yield 480,000 copies.

The term “copy” when used in reference to a first nucleic acid moleculeis intended to mean a second nucleic acid molecule having the samesequence as the first nucleic acid or the complementary sequence of thenucleic acid. The nucleic acids can be single stranded or doublestranded. For example, a single stranded copy can have the same sequenceof a single stranded template, a single stranded copy can have thecomplementary sequence of a single stranded template, a double strandedcopy can include the same sequence and the complementary sequence (i.e.two strands) of a single stranded template, or a double stranded copycan include the same sequences as a double stranded template. Similarly,the term “copy” when used in reference to a nucleic acid sequence meansthe same sequence or the complementary sequence.

As used herein, the terms “polynucleotide”, “oligonucleotide” or“nucleic acid” can refer to deoxyribonucleic acid (DNA), ribonucleicacid (RNA) or analogues of either DNA or RNA made, for example, fromnucleotide analogues. The terms “polynucleotide”, “oligonucleotide” or“nucleic acid” are applicable to single stranded (such as sense orantisense) and double stranded molecules. The terms “polynucleotide”,“oligonucleotide” or “nucleic acid” as used herein also encompass cDNA,that is complementary or copy DNA produced from an RNA template, forexample by the action of reverse transcriptase.

Single stranded polynucleotide molecules useful in a method orcomposition of the invention may have originated in single-strandedform, as DNA or RNA or may have originated in double-stranded DNA(dsDNA) form (e.g. genomic DNA fragments, PCR and amplification productsand the like). Thus a single stranded polynucleotide may be the sense orantisense strand of a polynucleotide duplex. Methods of preparation ofsingle stranded polynucleotide molecules suitable for use in the methodof the invention using standard techniques are well known in the art.

The term “immobilized” or “bound” as used herein is intended toencompass direct or indirect, covalent or non-covalent attachment,unless indicated otherwise, either explicitly or by context. In certainembodiments of the invention covalent attachment may be preferred, butgenerally all that is required is that the molecules (e.g. nucleicacids) remain immobilized or attached to a support under conditions inwhich it is intended to use the support, for example in applicationsrequiring nucleic acid amplification and/or sequencing.

In many embodiments of the invention, amplification primers for solidphase amplification are immobilized by covalent attachment to a solidsupport at or near the 5′ end of the primer, leaving thetemplate-specific portion of the primer free to anneal to its cognatetemplate and the 3′ hydroxyl group free to function in primer extension.The chosen attachment chemistry will depend on the nature of the solidsupport, and any functionalization or derivitization applied to it. Theprimer itself may include a moiety, which may be a non-nucleotidechemical modification to facilitate attachment. In particularembodiments the primer may include a sulphur containing nucleophile suchas phosphothioate or thiophosphate at the 5′ end. In the case of solidsupported polyacrylamide hydrogels, this nucleophile may bind to abromoacetamide group present in the hydrogel. In a preferred embodimentthe means of attaching the primers to the solid support is via 5′phosphothioate attachment to a hydrogel comprised of polymerisedacrylamide and N-(5-bromoacetamidylpentyl) acrylamide (BRAPA). Such anarrangement is described more fully in co-pending application WO05/065814, whose contents are incorporated herein by reference.

Single stranded template polynucleotide molecules may be attached to asolid support via hybridization to immobilized primers, or alternativelythe single stranded polynucleotide molecules may also be directlyattached to the solid support at or near the 5′ end. The chosenattachment chemistry will depend on the nature of the solid support, andany functionalization or derivitization applied to it. The singlestranded polynucleotide molecule itself may include a moiety, which maybe a non-nucleotide chemical modification to facilitate attachment. Inparticular embodiments a single stranded polynucleotide molecule mayinclude a sulphur containing nucleophile such as phosphorothioate orthiophosphate at the 5′ end. In the case of solid supportedpolyacrylamide hydrogels, this nucleophile can also bind to thebromoacetamide groups present in the hydrogel. In one embodiment themeans of attaching the single stranded polynucleotide molecule to thesolid support is via 5′ phosphorothioate attachment to a hydrogelcomprised of polymerised acrylamide and N-(5-bromoacetamidylpentyl)acrylamide (BRAPA). Such an arrangement is described more fully inco-pending application WO 05/065814, whose contents are incorporatedherein by reference.

The term “solid support” as used herein refers to any surface, inertsubstrate or matrix to which nucleic acids can be attached such as, forexample, beads, including latex or dextran beads, a surface, such as apolystyrene or polypropylene surface, polyacrylamide gel, gold surfaces,glass surfaces and silicon wafers. The solid support may be a glasssurface. The solid support may be a planar surface, although theinvention also works on beads which are moved between containers ofdifferent buffers, or beads arrayed on a planar surface. The solidsupport can be a flow cell, resin, gel, bead, well, column, chip,membrane, matrix, plate or filter.

In certain embodiments the solid support may comprise an inert substrateor matrix which has been “functionalized”, for example by theapplication of a layer or coating of an intermediate material comprisingreactive groups which permit covalent attachment to molecules such aspolynucleotides. By way of non-limiting example such supports mayinclude polyacrylamide hydrogels supported on an inert substrate such asglass. In such embodiments the molecules (e.g. polynucleotides) may bedirectly covalently attached to the intermediate material (e.g. thehydrogel) but the intermediate material may itself be non-covalentlyattached to the substrate or matrix (e.g. the glass substrate). Such anarrangement is described more fully in co-pending application WO05/065814, whose contents are included herein by reference.

Primer oligonucleotides or primers are polynucleotide sequences that arecapable of annealing specifically to one or more single strandedpolynucleotide template to be amplified under conditions encountered inthe primer annealing step of each cycle of an amplification reaction.Generally amplification reactions can use at least two amplificationprimers, often denoted “forward” and “reverse” primers. In certainembodiments the forward and reverse primers may be identical. Theforward primer oligonucleotides can include a “template-specificportion”, being a sequence of nucleotides capable of annealing to aprimer-binding sequence in at least one strand of the molecule to beamplified. Reverse primer oligonucleotides can include a templatespecific portion capable of annealing to the complement of the strand towhich the forward primer anneals during the annealing step. Generallyprimer oligonucleotides are single stranded polynucleotide structures.They may also contain a mixture of natural and non-natural bases andalso natural and non-natural backbone linkages, provided that anynon-natural modifications do not preclude function as a primer—thatbeing defined as the ability to anneal to a template polynucleotidestrand during conditions of the amplification reaction and to act as aninitiation point for synthesis of a new polynucleotide strandcomplementary to the template strand.

Primers may additionally comprise non-nucleotide chemical modifications,again provided that such modifications do not permanently prevent primerfunction. Chemical modifications may, for example, facilitate covalentattachment of the primer to a solid support. Certain chemicalmodifications may themselves improve the function of the molecule as aprimer, or may provide some other useful functionality, such asproviding a site for cleavage to enable the primer (or an extendedpolynucleotide strand derived therefrom) to be cleaved from a solidsupport.

Although the invention may encompass solid-phase amplification methods,in which only one amplification primer is immobilized on a solid support(the other primer usually being present in free solution), in aparticular embodiment, the solid support may be provided with both theforward and reverse primers immobilized. In practice there can be aplurality of identical forward primers and/or a plurality of identicalreverse primers immobilized on the solid support, for example, inembodiments wherein the amplification process utilizes an excess ofprimers to sustain amplification. Thus references herein to forward andreverse primers are to be interpreted accordingly as encompassing aplurality of such primers unless the context indicates otherwise.

“Solid-phase amplification” as used herein refers to any nucleic acidamplification reaction carried out on or in association with a solidsupport such that all or a portion of the amplified products areimmobilized on the solid support. In particular, the term encompassessolid phase amplification reactions analogous to standard solution phasePCR except that one or both of the forward and reverse amplificationprimers is/are immobilized on the solid support.

Primer oligonucleotides and single stranded polynucleotide moleculesthat have been immobilized on a solid support at a desired density canbe used to generate extension products by carrying out an appropriatenumber of cycles of amplification on the covalently bound singlestranded polynucleotide molecules so that each colony, or clustercomprises multiple copies of the original immobilized single strandedpolynucleotide molecule (and its complementary sequence). One cycle ofamplification can include steps of hybridization, extension anddenaturation. Such steps are generally comparable with the steps ofhybridization, extension and denaturation of PCR.

In embodiments utilizing solid phase amplification, suitable conditionscan be applied to a single stranded polynucleotide molecule and aplurality of immobilized primer oligonucleotides such that sequence Z atthe 3′ end of the single stranded polynucleotide molecule hybridizes toa primer oligonucleotide sequence X to form a complex wherein, theprimer oligonucleotide hybridizes to the single stranded template tocreate a “bridge” structure. Suitable conditions such as neutralizingand/or hybridizing buffers are well known in the art (See Sambrook etal., Molecular Cloning, A Laboratory Manual, 3^(rd) Ed, Cold SpringHarbor Laboratory Press, NY;

Current Protocols, eds Ausubel et al.). The neutralizing and/orhybridizing buffer may then be removed. One suitable hybridizationbuffer is referred to as “amplification pre-mix”, and contains 2 MBetaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2 mM Magnesium sulfate,0.1% Triton, 1.3% DMSO, pH 8.8.

By applying suitable conditions, an extension reaction can be performedfor a complex formed between immobilized primer and single strandedpolynucleotide template. The primer oligonucleotide of the complex canbe extended by sequential addition of nucleotides to generate anextension product complementary to the single stranded polynucleotidemolecule.

Examples of enzymes with polymerase activity which can be used in thepresent invention are DNA polymerase (Klenow fragment, T4 DNApolymerase, Bst polymerase), heat-stable DNA polymerases from a varietyof thermostable bacteria (such as Taq, VENT, Pfu, Tfl, Phusion DNApolymerases) as well as their genetically modified derivatives (TaqGold,VENTexo, Pfu exo). A combination of RNA polymerase and reversetranscriptase can also be used to generate the extension products. Auseful polymerase enzyme can have strand displacement activity. Thepolymerase enzyme can be active at a pH of about 7 to about 9,particularly pH 7.9 to pH 8.8. The nucleoside triphosphate moleculesused can be deoxyribonucleotide triphosphates, for example dATP, dTTP,dCTP, dGTP, or they can be ribonucleoside triphosphates for example ATP,UTP, CTP, GTP. The nucleoside triphosphate molecules may be naturally ornon-naturally occurring. An amplification reaction may also containadditives such as DMSO and or Betaine, for example, to normalise themelting temperatures of the different sequences in the template strands.A suitable solution for initial cycles of extension is referred to as“amplification mix” and contains 2 M betaine, 20 mM Tris, 10 mM AmmoniumSulfate, 2 mM Magnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8 plus 200pM dNTPs and 80 units/mL of Bst polymerase.

The denaturation can be carried out using heat or by using a denaturingbuffer. Suitable denaturing buffers are well known in the art (SeeSambrook et al., Molecular Cloning, A Laboratory Manual, 3^(rd) Ed, ColdSpring Harbor Laboratory Press, NY; Current Protocols, eds. Ausubel etal.). By way of example it is known that alterations in pH and low ionicstrength solutions can denature nucleic acids at substantiallyisothermal temperatures. Formamide and urea can be used fordenaturation. In a particular embodiment the concentration of formamideis 50% or more, and may be used neat. Such conditions result indenaturation of double stranded nucleic acid molecules to singlestranded nucleic acid molecules. Alternatively or additionally, thestrands may be separated by treatment with a solution of very low salt(for example less than 0.1 mM cationic conditions) and high pH (>12) orby using a chaotropic salt (e.g. guanidinium hydrochloride). In aparticular embodiment, a strong base may be used. A strong base is abasic chemical compound that is able to deprotonate very weak acids inan acid base reaction. The strength of a base is indicated by its pK_(b)value. Compounds with a pK_(b) value of less than about 1 are calledstrong bases and are well known to a skilled practitioner. In aparticular embodiment the strong base is Sodium Hydroxide (NaOH)solution used at a concentration of from 0.05 M to 0.25 M. Moreparticularly NaOH is used at a concentration of 0.1 M.

It may be advantageous to perform optional washing steps in betweensteps of an amplification method. For example, an extension bufferwithout polymerase enzyme with or without dNTPs could be applied to asolid support upon which amplification is being carried out and it canbe applied before being removed and replaced with complete extensionbuffer (extension buffer that includes all necessary components forextension to proceed).

Multiple cycles of amplification on a solid surface under conditionsexemplified above can result in a nucleic acid colony or “cluster”comprising multiple immobilized copies of a particular single strandedpolynucleotide sequence and its complementary sequence. Initialimmobilization of a single stranded polynucleotide molecule underconditions exemplified herein can result in the single strandedpolynucleotide molecule only hybridizing with primer oligonucleotideslocated at a distance within the total length of the single strandedpolynucleotide molecule. Thus, the boundary of the nucleic acid colonyor cluster formed can be limited to a relatively local area, namely thearea in which the initial single stranded polynucleotide molecule wasimmobilized. If conditions are used wherein the templates and thecomplementary copies thereof remain immobilized throughout the wholeamplification process, then the templates do not become intermingledother than by becoming large enough to overlap on the surface. Inparticular embodiments, there is no non-immobilized nucleic acid duringany part of the amplification process, and thus the templates cannotdiffuse and initiate further clusters elsewhere on the surface.

Hybridization, extension and denaturation steps of an amplificationmethod set forth herein may all be carried out at the same,substantially isothermal temperature. Preferably the temperature is from37° C. to about 75° C., depending on the choice of enzyme, morepreferably from 50° C. to 70° C., yet more preferably from 60° C. to 65°C. for Bst polymerase. In a particular embodiment the substantiallyisothermal temperature may be around the melting temperature of theoligonucleotide primer(s). Methods of calculating appropriate meltingtemperatures are known in the art. For example the annealing temperaturemay be about 5° C. below the melting temperature (Tm) of theoligonucleotide primers. In yet another particular embodiment thesubstantially isothermal temperature may be determined empirically. Thetemperature can be that at which the oligonucleotide displays greatestspecificity for the primer binding site whilst reducing non-specificbinding.

The term “common sequence,” when used in reference to a collection ofnucleic acid molecules, means a sequence that is the same for all of thenucleic acids in the collection. The nucleic acids in the collection canhave a region of common sequence despite the presence of at least oneother region in each of the nucleic acids that differs between thenucleic acids in the collection. As exemplified by the embodiments setforth above, all templates within a 5′ and 3′ modified library cancontain regions of common sequence Y and Z at (or proximal to) their 5′and 3′ ends, particularly wherein the common sequence at the 5′ end ofeach individual template in the library is not identical and not fullycomplementary to the common sequence at the 3′ end of said template. Theterm “library” refers to a collection or plurality of template moleculeswhich can share common sequences at their 5′ ends and common sequencesat their 3′ ends. Use of the term “5′ and 3′ modified library” to referto a collection or plurality of template molecules should not be takento imply that the templates making up the library are derived from aparticular source. By way of example, a “5′ and 3′ modified library” caninclude individual templates within the library that have the samenucleotide sequence or that have different nucleotide sequences.Furthermore, the templates can, but need not be related in terms ofsequence and/or source.

In various embodiments the invention can encompass use of so-called“mono-template” libraries, which comprise multiple copies of a singletype of template molecule, each having common sequences at their 5′ endsand their 3′ ends, as well as “complex” libraries wherein many, if notall, of the individual template molecules comprise different targetsequences, although all share common sequences at their 5′ ends and 3′ends. Such complex template libraries may be prepared from a complexmixture of target polynucleotides such as (but not limited to) randomgenomic DNA fragments, cDNA libraries etc. The invention may also beused to amplify “complex” libraries formed by mixing together severalindividual “mono-template” libraries, each of which has been preparedseparately starting from a single type of target molecule (i.e., amono-template). In particular embodiments more than 50%, or more than60%, or more than 70%, or more than 80%, or more than 90%, or more than95% of the individual polynucleotide templates in a complex library maycomprise different target sequences, although all templates in a givenlibrary can share a common sequence at their 5′ ends and a commonsequence at their 3′ ends.

Additives described herein are related at least for their ability tonormalize amplification of nucleic acid templates of differentsequences. The methods optionally include the use of differentconcentrations of nucleotides and/or nucleotide analogs as describedherein. The additives may be, for example, ethylene glycol, polyethyleneglycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide,7-deaza-GTP, acetamide, tetramethyl ammonium chloride (TMACI), salt orBetaine. For example, Betaine (carboxymethyl trimethyl ammonium((CH₃)₃N⁺CH₂COO⁻)) may be added to the amplification mix in order tonormalize the amplification of different template sequences. Optionally,a combination of Betaine and DMSO or a combination of Betaine, DMSO and7-deaza-dGTP is used. In particular, concentrations of Betaine may beabove 2 Molar (M), for example, between 2 and 5 M, between 2.5 and 4 Mor between 2.75 and 3.75 M.

Description

The present invention provides methods of reducing density-dependent GCbias in isothermal bridge amplification used for cluster generation. Inone embodiment, the method of the invention uses the addition of anadditive to a first solution in an isothermal bridge amplificationprotocol. In one example, the first solution in the bridge amplificationprotocol is a formamide solution used to denature dsDNA. In oneembodiment, the first solution further comprises EDTA. In one example,the additive is a mixture of dNTPs. In another example, the additive isa single dNTP (i.e., dATP, dCTP, dGTP, or dTTP). In yet another example,the additive is 10 mM trisodium citrate. In one embodiment, the additiveis 1 mM trisodium citrate. In one embodiment, the additive is less than1 mM trisodium citrate.

In another embodiment, the method of the invention uses the addition ofan additive to a second solution in an isothermal bridge amplificationprotocol. In one example, the second solution in the bridgeamplification protocol is a pre-mix solution. In one example, theadditive is a mixture of dNTPs. In another example, the second solutionin the bridge amplification protocol is water. In this example, watermay be used alone or with the addition of dNTPs, trisodium citrateand/or other additive. Replacing the pre-mix solution, which containssalts and buffers, with water enhances amplification of GC-richtemplates. An isothermal bridge amplification protocol for clustergeneration on a flow cell typically includes repeated cycles ofdenaturation, annealing, and extension. FIG. 1 illustrates a flowdiagram of an example of an isothermal bridge amplification protocol 100for cluster amplification on a flow cell surface. In one example, bridgeamplification protocol 100 is performed at about 60° C. and is repeatedany number of times (e.g., typically 35 times) to generate a clonalpopulation of identical DNA strands for each seeded DNA template. Forexample, the bridge amplification protocol may be repeated for one tofifty, one to twenty-five, one to fifteen, or one to ten cycles ofamplification. Bridge amplification protocol 100 includes, but is notlimited to, the following steps.

At a step 110, a first solution, e.g., formamide (e.g., about 28 μL), ispumped through each lane of the flow cell seeded with DNA template. Theformamide denatures the double-stranded cluster DNA on the flow cell tossDNA.

At a step 115, the formamide is removed by pumping a second solution,e.g., a solution of cluster pre-mix (e.g., about 28 μL), through eachlane of the flow cell. In one example, the pre-mix solution is astandard pre-mix solution comprising 20 mM Tris-HCl pH 8.8 @ 25° C., 10mM ammonium sulfate, 2 mM Mg sulfate, 0.1% Triton X-100, 1.3% DMSO, and2 M Betaine. Upon removal of formamide by washing with the pre-mixsolution, the denatured DNA strands bridge over and anneal tooligonucleotide primers bound to the flow cell surface.

At a step 120, a third solution, e.g., a solution of Bst mix (e.g.,about 36 μL), is pumped into each lane of the flow cell. The Bst mixcontains dNTPs and Bst DNA polymerase. In one example, a standard Bstmix solution is 20 mM Tris-HCl pH 8.8 @ 25° C., 10 mM ammonium sulfate,2 mM Mg sulfate, 0.1% Triton X-100, 1.3% DMSO, 2 M Betaine, 200 μM ofeach nucleotide, and 79 or 80 U/mL Bst polymerase. In another example,the Bst mix is an ammonium sulfate-free (NH₄-free) Bst mix of 20 mMTris-HCl pH 8.8 @ 25° C., 2 mM Mg sulfate, 0.1% Triton X-100, 1.3% DMSO,2 M Betaine, 200 μM of each nucleotide, and 79 or 80 U/mL Bstpolymerase. In the presence of the Bst mix, the surface-boundoligonucleotide primers that are hybridized to template molecules arefully extended.

Bridge amplification protocol 100 may be repeated any number of times.From a seeding site, a DNA molecule generates a cluster that isincreased in size at every amplification cycle. However, for anyparticular insert length (DNA template length), cluster size andintensity may be affected by the GC composition of the insert. Forexample, DNA templates that contain a high proportion of A and T (or Tand U for RNA) nucleotides tend to produce larger brighter clusterscompared to templates that have a higher proportion of G and Cnucleotides.

FIGS. 2A and 2B show a plot 200 of cluster size and a plot 250 ofcluster intensity, respectively, of clusters generated frommonotemplates with different GC composition. A monotemplate may bedefined as a single sequencing-ready template sequence (e.g., P5 primer,sequencing primer, template insert, and P7 primer) with a certain insertsize. In this example, monotemplates CT151 (31% GC), CT161 (50% GC),CT159 (55% GC), CT152 (64% GC), and CT180 (80% GC) with an insert of 400bp in size were seeded in different lanes of the same flow cell. After afirst extension, clusters were generated using 35 cycles of isothermalamplification. The flow cell was then stained with SYBR® Green and tworepresentative tiles for each lane were imaged using a microscope.Cluster size and intensities were determined using image analysissoftware Firecrest.

FIG. 3 shows a screenshot 300 of a cluster image showing GC-richclusters and AT-rich clusters. In this example, four monotemplates wereseeded at relatively low density. Clusters were generated byover-amplifying each molecule in order to generate larger clusters.After linearization, 3′ end blocking with ddNTPs, and hybridization ofthe sequencing primer, a first bridge amplification cycle was performed.A 3-color overlay image was then produced. Of the four monotemplatesseeded, three were AT-rich (clusters from the third AT rich monotemplateare not shown) and one was GC rich (80% GC). The clusters are labelledin the grayscale image as AT rich “G” and “B”, and GC rich “R”. Becauseof contact inhibition, clusters do not overlap with one another; theystop growing when they come into contact. In the current amplificationprotocol, AT rich templates tend to produce large clusters, whereas GCrich templates give origin to smaller clusters. As shown in FIG. 3, GCrich clusters (R) have been squeezed between AT rich clusters (G and B)and their growth has been restricted by the faster growing AT richclusters.

A decrease in cluster amplification efficiency of GC-rich clusters maybe due to DNA damage caused by cluster amplification reagents. When aDNA strand is damaged (e.g., either broken phosphate-sugar backbone ordamaged bases that stall primer extension) it may not act as template insubsequent amplification cycles and consequently, less efficient clusteramplification (i.e. smaller dimmer clusters) may be observed.

Modification of cluster amplification reagents by the addition ofadditives and/or the elimination of some components of the standardamplification reagent solutions may be used to ameliorate the DNA damageeffects. This may be achieved by reduction of DNA damage by formamide byusing an additive in the formamide solution, pre-mix solution, or both.Further, lowering the salt concentration may boost GC rich clustergrowth through a mechanism that does not involve DNA damage. Theseresults were surprising, It was previously unrecognised that formamidewas causing DNA damage.

FIG. 4 illustrates a flow diagram of an example of a DNA damage assay400 for evaluating the effect of cluster amplification reagents and/orthe addition of additives on cluster generation during isothermal bridgeamplification. FIG. 5 shows pictorially the steps of DNA damage assay400 of FIG. 4. Method 400 includes, but is not limited to, the followingsteps.

At a step 410, template DNA molecules are seeded onto the flow cell andhybridized to surface-bound oligonucleotide primers. This step is alsoshown pictorially in FIG. 5.

At a step 415, the template DNA molecules are copied (first extension)using DNA polymerase (e.g., Taq DNA polymerase or Phusion DNApolymerase) and the seeded template strand is then removed by flowing0.1 M NaOH through the flow cell (NaOH will also denature the DNApolymerase). Single stranded DNA molecules remain covalently bound tothe flow cell surface. This step is also shown pictorially in FIG. 5.

At a step 420, single stranded DNA molecules covalently bound to theflow cell surface are subjected to a treatment of “mock” isothermalamplification. The mock isothermal amplification comprises cycling thereagents that are used in cluster amplification (e.g., formamide,pre-mix, and amplification mix with the omission of Bst DNA polymeraseto avoid DNA amplification during treatment). In one example, the mockisothermal amplification comprises flowing formamide through a lane onthe flow cell, removing the formamide by flowing pre-mix through thelane of the flow cell, and flowing a pre-mix containing dNTPs onto theflow cell lane. Included on the flow cell is a control lane which servesas negative control (no DNA damage). For the control lane, a wash buffer(0.3×SSC containing 0.1% Tween-20) is continuously flowed through thelane; the wash buffer is non-DNA damaging. This step is also shownpictorially in FIG. 5.

At a step 425, all lanes are rinsed with wash buffer and then subjectedto isothermal amplification. Only non-damaged molecules can act as atemplate during this isothermal amplification step and therefore lead tocluster formation. Damaged DNA molecules or snapped molecules will notbe amplified. This step is also shown pictorially in FIG. 5.

At a step 430, clusters are visualized by staining with a DNA bindingdye (e.g., SYBR® Green) and imaged (e.g., three tiles for each lane)using a microscope and a camera. Image analysis software (e.g.,Firecrest software) is used to determine numbers of clusters. Bycounting the number of clusters from each lane, it is possible to inferhow many molecules have been damaged by a particular treatment.

Potential DNA damage caused by standard isothermal amplificationreagents were evaluated using DNA damage assay 400 of FIG. 4 and seededmonotemplates. In one example, a CT180 monotemplate may be used in DNAdamage assay 400 to evaluate potential DNA damage caused by isothermalamplification reagents. The CT180 monotemplate comprises 80% GC (i.e.,it is GC rich) and has an insert size of 400 bp.

FIG. 6 shows a bar graph 600 of the percent of non-damaged DNA moleculesin samples treated with formamide, pre-mix, and/or pre-mix plus dNTPsand evaluated using DNA damage assay 400 of FIG. 4. In this experiment,monotemplate CT180 was seeded at 1 pM. After first strand extension withTaq DNA polymerase and template denaturation with NaOH, single DNAmolecules were treated with 26 cycles of mock isothermal amplification(step 420 of DNA damage assay 400 of FIG. 4) using a first solution of28 μL of formamide, a second solution of 28 μL of pre-mix without orwith the addition of dNTPs, and a third solution of 36 μL of pre-mixwith the addition of dNTPs. Each bar on the graph represents a lane onthe flow cell. The control lane (first graph bar) was treated with washbuffer for the entire duration of the treatment (26 cycles). Clusternumbers for each treated lane (graph bars 2 and 3) on the flow cell aredivided by the cluster number from the control lane and are expressed asa percentage of the control (reference) lane. Lane 2, which was treatedwith 26 cycles of mock isothermal amplification using the standardconditions (formamide/pre-mix/premix with dNTPs) shows a significantdecrease in the number of clusters demonstrating that the isothermalamplification reagents cause DNA damage (a primer-density assayexperiment showed that there was no significant loss of surface oroligonucleotides from the surface, data not shown). When dNTPs wereincluded in the pre-mix solution (pre-mix/dNTPs) that was pumpedimmediately after formamide, DNA damage was less severe (compare lanes 2and 3). These results suggest that dNTPs are protecting DNA from damagecaused by cluster amplification reagents (e.g., formamide).

FIG. 7 shows a bar graph 700 of the percent of non-damaged DNA moleculesin samples treated with formamide±dNTPs, pre-mix±dNTPs, or water±dNTPsand evaluated using DNA damage assay 400 of FIG. 4. In this experiment,monotemplate CT180 was seeded at 1 pM. After first strand extension withTaq DNA polymerase and template denaturation with NaOH, single DNAmolecules were treated with 26 cycles of mock isothermal amplification(step 420 of DNA damage assay 400 of FIG. 4) using three solutions. Thefirst solution was either 28 μL of wash buffer (WB), formamide (F), orformamide plus dNTPs (F/nt). The second solution was either 28 μL ofwash buffer (WB), formamide (F), pre-mix, pre-mix plus dNTPs(pre-mix/nt), water (H₂O), or water plus dNTPs (H₂O/nt). The thirdsolution was 36 μL of wash buffer (WB) or pre-mix/nt. dNTPs were used ata final concentration of 200 μM each. Each bar on the graph represents alane of the flow cell. The data show the protective effect of dNTPs whenpresent in the pre-mix solution (comparing lanes 3 and 4), in formamide(comparing lanes 4 and 5), and in water (comparing lanes 6 and 7). Lane8 also shows that the wash buffer is also protecting the DNA moleculesfrom damage caused by formamide (compare lanes 2 and 8).

The effect of dNTP concentration on reducing formamide-induced DNAdamage was evaluated in a titration experiment in which different amountof dNTPs were added to formamide. FIG. 8 shows a dose response curve 800of the effect of dNTP concentration in formamide on cluster numberduring isothermal mock amplification. In this experiment, CT180 wasseeded at 0.6 pM. First strand extension was performed using Taq DNApolymerase followed by NaOH denaturation. Single molecules weresubsequently treated with 26 cycles of mock isothermal amplificationaccording to DNA damage assay 400 of FIG. 4 using 28 μL of a firstsolution of formamide with different dNTP concentrations, 28 μL of asecond solution of water with the same concentrations of dNTPs, and 36μL of a third solution of pre-mix with the same concentrations of dNTPs.After the mock isothermal amplification, clonal populations of molecules(clusters) were generated with 26 cycles of isothermal amplificationusing 28 μL of formamide followed by 64 μL of Bst mix in each cycle (nopre-mix was used). The results show a dose response of DNA damageabrogation with higher concentrations of dNTPs being more effective atpreventing DNA damage.

The effect of individual dNTPs on reducing formamide-induced DNA damagewas evaluated using DNA damage assay 400 of FIG. 4. FIG. 9 shows a bargraph 900 of the effectiveness of each dNTP to prevent DNA damage whenused as a formamide additive. In this experiment, CT180 was seeded at0.6 pM. First strand extension was performed using Taq DNA polymerasefollowed by NaOH denaturation. Single molecules were subsequentlytreated with either wash buffer (WB), formamide (F), or formamide plusdATP, dCTP, dGTP, dTTP, or a dNTP mix for 80 minutes at 60° C. (a timeperiod that is similar to the duration of 26 cycles of isothermalamplification). After treatment, cluster amplification was performedusing 26 isothermal cycles of 28 μL formamide followed by 64 μL of Bstmix for each cycle. The final concentration of the individualnucleotides was 800 μM. The concentration of each nucleotide in the dNTPmix was 200 μM. The data show that all four nucleotides are equallyeffective at protecting DNA from damage. The results also show thatdNTPs prevent DNA damage caused by formamide when this chemical ispumped constantly (instead of being cycled together with water andpre-mix).

To determine whether the addition of dNTPs may rescue clonalamplification when a relatively large volume of formamide is used in anisothermal amplification process, amplification was performed using 120μL of formamide with or without the addition of dNTPs. FIG. 10 showspanels of clusters 1000 generated by isothermal amplification usingformamide with and without added dNTPs. In this example, two templateswere used, CT180 (a GC-rich template) and CT3164 (an AT-rich template).The left-hand panels, from top to bottom, represent lanes 1 through 4 ofthe flow cell. The right-hand panels, from top to bottom, representlanes 5 through 8 of the flow cell. Lanes 1 through 4 on the flow cellwere seeded with 0.6 pM of CT180; lanes 5 through 8 on the flow cellwere seeded with 0.6 pM of CT3164. After the first strand extensionusing Taq DNA polymerase, followed by NaOH denaturation, clusteramplification was performed using 26 isothermal amplification cycles of120 μL of formamide (a relatively large volume compared to the standard28 μL of formamide), followed by 28 μL of water, and 36 μL of standardBst mix. Clusters were visualized by staining with SYBR® Green andimaged. A representative tile from each lane is shown in FIG. 9. Thedata show that while both the CT180 and CT3164 templates fail to beamplified in an isothermal amplification process using a relativelylarge volume of formamide, cluster amplification is rescued by theaddition of dNTPs into the formamide solution.

FIG. 11 shows a data table 1100 of sequencing metrics for a sequencingrun evaluating the “dNTPs” method and the “water” method. The dNTPsmethod includes the addition of dNTPs to both a first solution offormamide and a second solution of water. The water method replaces asecond solution of pre-mix with water. In this experiment CT4008(standard BCG library, GC rich genome; average insert size of 300 bp)was seeded at either low density (1.5 pM) or high density (7.5 pM).After standard first strand extension with Phusion DNA polymerase,clusters were amplified with 26 cycles of isothermal amplification usingdifferent conditions: No—premix=28 μL formamide and 64 μL Bst mix;pre-mix=28 μL formamide, 28 μL pre-mix, and 36 μL Bst mix; watermethod=28 μL formamide, 28 μL water, and 36 μL Bst mix; dNTPs method=28μL formamide containing 200 μM each nucleotide, 28 μL water containing200 μM each nucleotide, and 36 μL Bst mix. This run was analyzed withPipeline 1.8. The flow cell was sequenced with 36 cycles of SBS using95G chemistry on a Genome Analyzer IIx.

For the BCG genome, there was a significant increase in terms ofclusters passing filter (see “% PF clusters” column data table 1100) forboth the “water” and “dNTPs” methods compared to the no-premix and thestandard amplification methods (labelled “pre-mix” in the Figure). Thebrightest clusters were obtained with the “dNTPs method” (compare lanes6 and 8 with lanes 4 and 7).

FIGS. 12A and 12B show curves 1200 of GC bias and curves 1250 of GC biasnormalized to low density lane 1, respectively, of the sequencing rundescribed with reference to FIG. 11.

FIG. 13A shows a summary schematic diagram 1300 of bridge amplificationsolutions according to the methods of the invention, while FIG. 13Bshows a summary schematic diagram 1350 of bridge amplification solutionsaccording to a standard method. In one embodiment, trisodium citrate isused in a final concentration of 1 mM in formamide.

In another aspect of the present invention, solid phase amplificationcan be performed efficiently in a flow cell since it is a feature of theinvention that the primers, template and amplified (extension) productsmay all remain immobilized to the solid support during theamplification. Accordingly, an apparatus is provided that can allowimmobilized nucleic acids to be isothermally amplified. An apparatus mayalso include a source of reactants and detecting means for detecting asignal that may be generated once one or more reactants have beenapplied to the immobilized nucleic acid molecules. An apparatus may alsobe provided with a surface comprising immobilized nucleic acid moleculesin the form of colonies.

In one embodiment, an apparatus is provided comprising one or more ofthe following:

-   -   a) at least one inlet    -   b) means for immobilizing primers on a surface (although this is        not needed if immobilized primers are already provided);    -   c) means for substantially isothermal amplification of nucleic        acids (e.g. denaturing solution, hybridizing solution, extension        solution, wash solution(s));    -   d) at least one outlet; and    -   e) control means for coordinating the different steps required        for the method of the present invention.

In other embodiments, as a volume of a particular suitable solution incontact with a solid support is removed, it is replaced with a similarvolume of either the same or a different solution. Thus, solutionsapplied to a flow cell through an inlet can be removed via an outlet bya process of solution exchange.

Desirably, a means for detecting a signal has sufficient resolution toenable it to distinguish between and among signals generated fromdifferent colonies. Instruments that are useful for detecting afluorescent signal are described, for example, in WO 2007/123744, US2010/0111768 and U.S. Pat. No. 7,329,860, the contents of which areincorporated by reference herein in their entireties.

Apparatuses of the present invention are preferably provided inautomated form so that once they are activated, individual process stepscan be repeated automatically.

CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. The term “theinvention” or the like is used with reference to certain specificexamples of the many alternative aspects or embodiments of theapplicants' invention set forth in this specification, and neither itsuse nor its absence is intended to limit the scope of the applicants'invention or the scope of the claims. This specification is divided intosections for the convenience of the reader only. Headings should not beconstrued as limiting of the scope of the invention. The definitions areintended as a part of the description of the invention. It will beunderstood that various details of the present invention may be changedwithout departing from the scope of the present invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation.

The invention claimed is:
 1. A method for reducing density-dependent GCbias and/or nucleic acid damage in bridge amplification of adouble-stranded DNA template on a surface comprising: (a) denaturing thedouble-stranded DNA template with a first solution comprising formamideand at least one type of dNTP to produce single-stranded DNA templatestrands; (b) annealing the single-stranded DNA template strands tooligonucleotide primers bound to the surface; and (c) extending theoligonucleotide primers by replacing the first solution with a solutioncomprising a polymerase and a mixture of different dNTPs, whereby theoligonucleotide primers bound to the surface are fully extended; and (d)repeating steps (a)-(c) at least once; wherein density-dependent GC biasand/or nucleic acid damage in the amplification of the double-strandedDNA template is reduced.
 2. The method of claim 1, wherein firstsolution comprises at least one additive.
 3. The method of claim 2,wherein the additive comprises a chelating agent.
 4. The method of claim2, wherein the additive comprises at least one citrate.
 5. The method ofclaim 4, wherein the at least one citrate is selected from: monosodiumcitrate, disodium citrate, trisodium citrate, and potassium citrate. 6.The method of claim 2, wherein the additive comprises EDTA.
 7. Themethod of claim 2, wherein the additive comprises betaine.
 8. The methodof claim 2, wherein the additive comprises DMSO.
 9. The method of claim1, wherein a cluster of identical DNA strands is generated on thesurface.
 10. The method of claim 1, wherein the first solution isreplaced by a second solution after step (a) and before step (b),wherein the second solution comprises water and/or a pre-mix solutioncomprising one or more components selected from betaine, Tris, ammoniumsulfate, magnesium sulfate, polyethylene glycol tert-octylphenyl ether,and DMSO.
 11. The method of claim 10, wherein the second solutioncomprises less than about 100 mM of salt.
 12. The method of claim 11,wherein the second solution is substantially free from salt.
 13. Themethod of claim 1, wherein the solution comprising the polymerasecomprises less than about 100 mM of salt.
 14. The method of claim 13,wherein the solution comprising the polymerase is substantially freefrom any salt.